Impact of positive CD4 cells on event‐free survival in follicular lymphoma patients

Abstract Objective Previous results about prognostic value of CD4+ T cells in follicular lymphoma (FL) remain controversial. Methods Immunohistochemistry was used to examine expression of positive CD4 cells in 103 patients with FL 1‐3A. Early failure was described as failing to achieve event‐free survival (EFS) at 12 or 24 months. Results There were 49 (47.6%) male and 54 (52.4%) females, with a median age of 54 years. Compared to patients with <20% of positive CD4 cells, patients with ≥20% of positive CD4 cells exhibited a significant lower risk of early failure (2‐year EFS rate: 56.7% vs 73.5%, p = 0.047). When patients were stratified based on positive CD4 cell combined with FLIPI, the median EFS (p = 0.002) and median OS (p = 0.007) were significantly different. Conclusions This study demonstrated that higher expression of positive CD4 cells predicts lower risk of early failure in follicular lymphoma, and combination analysis of CD4 and FLIPI could better predict disease relapse and survival outcome.

Lymphoma International Prognostic Index (FLIPI) was established in the pre-rituximab era, thus, it exhibits limited accuracy in the current immunochemotherapy era. 5 The FLIPI-2 and PRIMA-PI mainly predicts PFS, but not OS. 6,7Lastly, the m7-FLIPI and 23-gene predictor used expensive and complicated sequencing and gene expression profiling, which, unfortunately, limited their clinical practice. 8,9merging evidences reported that the tumor microenvironment (TME) is critical for FL onset and progression.Whole-genome microarray of diagnostic FL biopsies uncovered that the patient genetic profile associated with desirable prognosis was enriched in T-cell gene expression.In contrast, the genetic profile linked to worse prognosis featured macrophages. 10Until now, the understanding of the FL TME is still not comprehensive because of the highly heterogeneous components of TME.Among various cell subsets, T cells are the most prevalent TME components in FL, including 10%-40% of naïve and memory CD4 T cells. 11In the past, the tumor-infiltrating CD4+ T cells were examined using numerous approaches of prognostic indicator analyses. 12,13However, the conclusions were inconsistent.Hence, in this investigation, we examined the expression and location of CD4 cells in the FL tumor tissue using immunohistochemistry (IHC).We also conducted survival analysis to elucidate whether positive CD4 cell expression can predict the early failure and overall survival of FL patients.

| Patients
For analysis, we recruited patients with newly diagnosed FL grade 1-3A.Patients with histologic grade 3B FL or transformation/composition of diffuse large B cell lymphoma (DLBCL) were excluded from this study.Diagnosis was based on the World Health Organization (WHO) histological guidelines, and was conducted by a highly skilled pathologist.Sufficient diagnostic formalinfixed, paraffin-embedded (FFPE) lymph node tissues were obtained to examine IHC expression.Cases with negative CD4 cell expression were excluded from analysis.Treatment regimens were guided by the Groupe d'Etude des Lymphomes Folliculaires (GELF), or National Comprehensive Cancer Network (NCCN) criteria. 14,15n terms of stage I-II patients, initial therapy included involved-site radiotherapy (ISRT) or ISRT+anti-CD20 monoclonal antibody±chemotherapy, surgery resection, or observation.In terms of stage III-IV patients, observation was recommended for patients with no indication for treatment, otherwise, systemic chemotherapy was selected.Blood cell counts, circulation biochemistry, computerized tomography scan, and bone marrow biopsy results of patients were collected.The patient response was evaluated based on conventional guidelines. 16All patients underwent follow-up to monitor disease progression/relapse, retreatment, transformation, and death.Moreover, all medical events were verified using medical records.This research received ethical approval from the Zhejiang Cancer Hospital, and obtained informed consents from all subjects prior to the initiation of the study.

| IHC staining
The paraffin-embedded lymph node blocks prior to treatment before treatment were cut into 4-μm sections and before application to 3-aminopropyltrioxysilane-coated slides.Subsequently, the specimen underwent dewaxing and blocking in a hydrogen peroxide/methanol mixture, followed by antigen retrieval via cooking the specimen in citrate in a pressure cooker.Next, the specimen underwent staining via the Vector Elite ABC kit (Ventana Medical Systems, Tucson, AZ), with subsequent diaminobenzidine chromogen staining (Biostat, Stockport, United Kingdom), as per kit directions.

| IHC analysis
Two histopathologists independently scored all IHC results, and obtained comparable results.Tissue microarrays (TMA) were stained with CD4 according to the manufacturers' recommendations.In short, the number of CD4 positive cells per high performance fortran (HPF) (×400 magnification) was counted, and its proportion was recorded.Based on the staining degree, the CD4 positive cellular densities were categorized as follows: −, none cells/HPF; +, weakly staining cells/HPF; ++, moderately positive cells/HPF; +++, strongly positive cells/HPF.The immunoarchitectural profiles of positive CD4 cell distributions were assessed in comparison to neoplastic follicles.The "follicular" profile was manifested by peri-and intrafollicular CD4 positive cell predominance.Alternately, the "diffuse" profile was defined by diffusely distributed cells, with no obvious association with follicles.

| Statistical analysis
Event-free survival (EFS) was described as the duration from diagnosis to disease progression, relapse, therapy initiation, transformation to aggressive lymphoma, or death due to any cause.The interval between diagnosis and treatment initiation for patients with indications for treatment was less than 3 months.Early failure was described as not achieving EFS at 24 months (EFS24) in patients who received treatment immediately after diagnosis, or not achieving EFS at 12 months (EFS12) in patients under observation.Overall survival (OS) was computed from the diagnosis date till the death from any cause date or date of last contact.All time-associated endpoints were predicted using Kaplan-Meier, and the log-rank test was employed to compare between risk groups, as well as estimate hazard ratios (HRs) and 95% confidence intervals (CI).Categorical variables were analyzed using Fisher's exact or χ 2 test, where appropriate, and continuous variables were analyzed via two-tailed paired t tests.Two-sided p < 0.05 was set as the significance threshold.Lastly, SPSS, version 26 was employed for all data analyses.

| Patients characteristics and treatment
Between April 2004 and December 2019, 103 patients with positive CD4 cellular expression, based on IHC, were recruited for analysis.The baseline clinical profile of these patients is presented in Table 1.There were 49 (47.6%)males and 54 (52.4%) females, with a median age of 54 year (range, 27-81 years) at diagnosis.A larger proportion of patients exhibited stage III-IV disease (66/103, 64.1%) and pathological grade 1-2 (63/103, 61.2%).

| Relationship between positive CD4 cells and clinical features
The relationship between positive CD4 cells and primary patient clinical characteristics are listed in Table 1 FLIPI-2, and PRIMA-PI.However, patients with low disease stage exhibited significantly more positive CD4 cells ≥20%, compared to patients with high disease stage (75.7% vs 50%, p = 0.011).

| DISCUSSION
Besides common clinical parameters, lymphomagenesis of FL are highly dependent on interactions with a specific and complex TME. 17The TME of FL is composed of follicular dendritic cells (FDCs), follicular regulatory T (Treg) cells, tumor-infiltrating T cells (TILs), lymphoma-associated macrophages (LAMs), and immune checkpoint-related immune cells. 11,18Among these components in FL TME, CD4+ TILs play an important role.They can impair the recruitment and activity of essential transduction proteins to the immunologic synapse. 19They also exhibit drastically dysfunctional motility, relative to healthy TILs from the reactive tonsils. 20Cytotoxic CD4 T cells can eliminate senescent fibroblasts, presenting their broad immune surveillance capability. 21Low CD4 expression or absence of intrafollicular memory CD4+ T cells can be helpful to predict patient risk for POD24. 22,23Therefore, we selected CD4 as a potential prognostic indicator in this study.
Most previous studies examining the relationship between CD4 expression and patient survival are controversial. 24Using IHC assessment, Abigail M Lee al. demonstrated that CD4+ stained cells were significantly more common in FL patients who survived for over 15 years, compared to their short-lived counterparts. 25In the Wahlin et al. study, a tissue microarray was generated from 70 FL biopsies, and cellular subsets were quantified via computerized image analysis.The results revealed that a large proportion of intrafollicular CD4+ cells, as well as elevated CD4 follicular/interfollicular ratios, are strongly correlated with worse prognosis in FL patients. 26imilarly, the Anna Maria study also employed tissue microarrays, multiplex immunofluorescence, and Shannon's entropy to test FL tissues in 127 patients, who received different treatments.Based on her results, the enhanced immune infiltrate population diversity, including CD4, is strongly associated with improved OS. 27 Yang et al evaluated 82 FL patient tumor tissues using Mass cytometry (CyTOF).When they analyzed patients with FL grade 1-2, they reported that increased T cells (including CD4+) significantly correlated with a favorable survival.But there is no similar prognostic significance in patients with FL grade 3. 28 Sayako Yuda et al. examined variables that influenced the treatment-free period in FL patients using a watch-and-wait approach.They reported that elevated interfollicular CD4+ quantities in the TME were a significant adverse factor in univariate analyses. 29However, other studies observed no prognostic value for CD4 at both the protein and mRNA levels. 30,31We speculate that patient selection and different testing or analytical methods may have affected the final results in the aforementioned publications.To better illustrate the impact of CD4 expression on patient survival, we merely focused on cases with a positive CD4 expression, and excluded cases without CD4 expression.Additionally, we utilized IHC to evaluate the CD4 expression within FL tumor tissues as IHC is the most widely employed technique in both clinical and pathologic practices, and it has the characteristics of convenience, simplicity, and repeatability.Another possible reason for the heterogeneity within the aforementioned results is the definition of the study end point.The treatment regimens for FL 1-3A were heterogeneous.A 'watch and wait' approach is a potential strategy for asymptomatic FL patients. 32However, some patients may experience disease progression or transformation, and may require immediate anti-tumor intervention.Similarly, some symptomatic FL patients can experience disease relapse after immunochemotherapy.The interval between the start of immunochemotherapy and disease relapse or the interval between disease diagnosis and first-line treatment onset is an essential indicator of early failure.Therefore, it is unreasonable to employ the same study endpoint to define the early failure events for all patients, including both early and advanced FL stages, as was performed in the aforementioned studies. 26,31In order to avoid this unreasonable issue, we defined early failure as the failure to achieve EFS12 for observed patient or failure to achieve EFS24 for treated patients.
Our results revealed that an elevated positive CD4 expression indicated a lower risk of early failure in FL patients.In terms of using FLIPI-2, PRIMA-PI, and FLIPI to predict patient EFS or OS, high-risk FLIPI was far superior in identifying patients with poor survival (44.1%) and high-risk of early failure (52.9%), even though the FLIPI scoring system was developed in the pre-rituximab era.When patients were classified by FLIPI and the positive proportion of CD4 cells, patients with FLIPI score 3-5 and positive CD4 cells <20% were significantly associated with early failure and inferior OS.
Prior studies revealed that elevated CD4+ infiltration is more prominent in patients without B symptoms, low performance status, and enhanced transformation risk. 26,33n addition to CD4 quantity, the location of positive CD4 cells is another essential factor in host response to FL. 34 The Toma et al study demonstrated that 98.4% of CD4 are present outside the neoplastic follicles. 33The spatial distribution of CD4-positive T-helper cells is known to be highly discriminatory in predicting early FL transformation. 35In this study, the positive CD4 cell expression profile primarily included perifollicular and diffuse profiles.Moreover, positive CD4 cells ≥20% occurred more commonly in patients with low disease stages than with high disease stages.However, the positive CD4 cell expression profile and intensity showed no impact on EFS12/24 or OS.
However, a critical question arises while analyzing these results.FL pathogenesis and prognosis are affected by various immune cell compositions working simultaneously in the TME, rather than being dictated by the action of an individual immune cell subset. 36Therefore, it is difficult to elucidate the entire function and role of the TME by examining a single cell or cytokine.Some modern technologies, including single-cell RNA sequencing, can identify previously unknown cell subsets and define their distribution patterns and expression degree, however, the clinical and biological significance of TME is not entirely clear. 13,37,38The important significance of our study is that by a simple and affordable testing method for a regular histopathological index, we can better screen patients with early failure or poor outcome at the time of diagnosis.

| CONCLUSION
This study demonstrated that elevated CD4 positive cell expression in FL patients has a lower risk of early failure.Hence, a combination of positive CD4 cells <20% with high risk FLIPI can better predict patients with early failure and poor OS.

F
I G U R E 4 Event-free survival (EFS) and overall survival (OS) in patients with FL based on positive CD4 cells combined with FLIPI risk model.
Patient and disease characteristics.
The expression, intensity, and distribution of positive CD4 cells in 103 follicular lymphoma patients.Event-free survival (EFS) and overall survival (OS) in patients with FL in relation to positive CD4 cells (<20% vs ≥20%).
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